Improved Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for the Rapid and Sensitive Detection of Yam mosaic virus

Yam (Dioscorea spp.) productivity is constrained significantly by the lack of a formal seed system. Vegetative propagation, through tuber setts as ‘seed’ yams, encourages the recycling of virus-infected planting materials, contributing to high virus incidence and yield losses. Efforts are ongoing to increase the production of high-quality seed yams in a formal seed system to reduce virus-induced yield losses and enhance the crop’s productivity and food security. Specific and sensitive diagnostic tests are imperative to prevent the multiplication of virus-infected materials contributing to a sustainable seed yam certification system. During routine indexing of yam accessions, discrepancies were observed between the results obtained from the reverse transcription loop-mediated isothermal amplification (RT-LAMP) test and those from reverse transcription polymerase chain reaction (RT-PCR); RT-LAMP failed to detect Yam mosaic virus (YMV) in some samples that tested positive by RT-PCR. This prompted the design of a new set of LAMP primers, YMV1-OPT primers. These primers detected as little as 0.1 fg/µL of purified RNA obtained from a YMV-infected plant, a sensitivity equivalent to that obtained with RT-PCR. RT-LAMP using YMV1-OPT primers is recommended for all future virus-indexing of seed yams for YMV, offering a rapid, sensitive, and cost-effective approach.


Introduction
Yam mosaic virus (YMV) is a prevalent virus of yam [1][2][3][4], an important staple food crop in many parts of the world [5]. It belongs to the genus Potyvirus and has a single-stranded, positive-sense RNA genome that is approximately 9.6 kb long and encodes a single large polyprotein, which is cleaved into smaller proteins [6][7][8][9]. YMV is widely distributed in tropical and subtropical yam-growing regions, particularly in West Africa, the West Indies, and the Caribbean [3,4,10]. It commonly infects D. rotundata, D. cayenensis-rotundata, and D. alata [3,11]. The virus is transmitted through vegetative propagation of infected yam materials or by aphid vectors in a non-persistent manner, causing various symptoms, including mosaic patterns on leaves, stunted growth, and reduced yields [3]. YMV has been reported to cause about 40% yield loss in yam fields [12,13] and hamper the exchange of valuable germplasm for the crop's improvement.
Yam plays a vital role in food security, income generation, and nutrition for smallholder farmers, especially in West Africa, which produces over 95% of the world's total yam production [3,5,14]. An infection with YMV in the field threatens the food security and livelihoods of West Africans. The absence of a formal seed yam certification system and farmers selecting small tubers from their harvest for planting the following season encourage the propagation of infected materials, which has been instrumental to the spread of YMV in yam-growing regions [15][16][17].
The use of virus-free planting materials is the most effective method to control the spread of viruses infecting yam [18,19]. Several methods have recently been developed to boost the production of virus-free seed yams, including single-node vine cuttings, tissue culture, hydroponics, and aeroponic systems [16,20,21]. The development of sensitive and cost-effective diagnostics is paramount to guarantee the production of virus-free seed yams for a sustainable formal seed system [22,23]. These diagnostics methods must address virus detection challenges, including false-negative results arising from reduced virus titre associated with clonally propagated crops [8]. Furthermore, the genomic variability of YMV makes the detection of all putative isolates/variants challenging [24].
Routine indexing of yam plants for YMV in our laboratories identified discrepancies between the RT-LAMP [23] and RT-PCR [25] tests. RT-LAMP gave false-negative results for some samples, which were confirmed positive for YMV by RT-PCR and Sanger sequencing. False-negative results could permit the multiplication of infected plant materials in the seed systems, discrediting the integrity of quality seeds distributed to farmers [19,28]. Further, it could pose severe challenges to plant health by spreading viruses or novel variants to new regions through the exchange of infected germplasms [28]. This prompted the development of a new set of LAMP primers which are described in this article and were found to increase not only the specificity but also the sensitivity of YMV detection compared to existing YMV LAMP primers [23].

Plant Material, Total RNA Extraction, and Crude Sample Preparation
Yam (D. rotundata and D. alata) leaf tissues used in this study were obtained from plants grown in glasshouses at the Natural Resources Institute (NRI), United Kingdom, the Centre for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI) in Ghana, and yam field surveys conducted in Benin, Cameroon, Togo, and Nigeria (Table 1). Total RNAs were extracted from leaf tissues using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, Saint Louis, MO, USA), according to the manufacturer's recommendations. The concentration and purity of extracted yam RNAs were measured using a NanoDrop 2000 spectrometer (ThermoScientific, Waltham, MA, USA).
The detection of YMV from crude extracts was carried out using the protocol described by Silva et al. [27]. One leaf disc was immersed in 300 µL of freshly prepared PEG buffer (6% w/v polyethylene glycol (PEG)−200 in 20 mM NaOH). The tubes were vortexed briefly and incubated for 5 min at room temperature. Crude extracts were used directly as templates in RT-LAMP assays.

Sample ID Collection Origin
Dioscorea spp.

The Detection of YMV by RT-PCR and Phylogenetic Analysis
The detection of YMV by RT-PCR was carried out using the primer pair YMV CP 1F and YMV UTR 1R (Table 2), which amplifies a 586 bp region comprising a partial coat protein (CP) gene and the 3' UTR region of the YMV genome [25]. The RNA quality was confirmed by amplifying the yam actin gene, as described by Silva et al. [22]. RT-PCR assays were set up as 20 µL reactions containing 0.2 µM of each primer (Sigma Aldrich), 0.25 mM of each dNTP (ThermoScientific), 1.25 U DreamTaq DNA Polymerase (ThermoScientific), 2.5 U AMV-reverse transcriptase (Promega, Madison, WI, USA), 1X DreamTaq Green Buffer containing 2 mM MgCl 2 (ThermoScientific), and 2 µL RNA as template. Thermal cycling conditions were 50 • C for 10 min, followed by 95 • C for 4.5 min, and 35 cycles of 95 • C for 30 s, 55 • C for 30 s, and 72 • C for 30 s, and a final extension of 72 • C for 10 min. RT-PCR products were analysed by electrophoresis on agarose gels [2% (w/v) agarose in Tris-borate-EDTA (0.5 × TBE) buffer (pH 8.0)] and viewed under UV light using a gel doc system (SynGene, Cambridge, UK). PCR products were purified and Sangersequenced by the Source BioScience sequencing service (Cambridge, UK). The nucleotide sequences generated from the PCR products were analysed and assembled using Geneious Prime ®® 2023.0.1 (Biomatters Ltd., Auckland, New Zealand). Sequences were used for similarity BLAST searches in the National Centre for Biotechnology Information (NCBI) GenBank databases. Thirty YMV coat protein (CP) sequences, representing YMV phylogenetic groups classified by Bousalem et al. [24] and Mendoza et al. [9], were downloaded from NCBI and aligned with 36 YMV CP sequences obtained from this study ( Table 3) to generate a percentage similarity matrix using Multiple Alignment using Fast Fourier Transform (MAFFT) v7.490 in Geneious Prime ®® 2023.0.1. The aligned sequences were used for phylogenetic analysis using the Neighbor-Joining (NJ) method in Molecular Evolutionary Genetics Analysis across Computing Platforms (MEGA X) v10.2.6 software [30]. The reliability of the tree branches was evaluated by bootstrap test in 1000 replicates.  Group*-YMV phylogenetic group following classification by Bousalem et al. [24], Mendoza et al. [9] and this study.

New LAMP Primer Design for YMV Detection
A multiple sequence alignment of 125 YMV CP sequences (downloaded from the NCBI GenBank database on 5 April 2021) was carried out using the Mafft Alignment v7.450 in Geneious Prime ®® 2021.1.1 (Biomatters Ltd., Auckland, New Zealand). A consensus sequence based on the alignment was used to design new LAMP primers using the Primer Explorer V5 software (http://primerexplorer.jp/e/) and visual adjustment of the primers' position to avoid mismatches.

Detection of YMV by RT-LAMP
The same RNAs analysed by RT-PCR were used as templates in RT-LAMP. Two sets of primers were used ( Table 2). Each RT-LAMP reaction was carried out in three replicates. The RT-LAMP assays were set up as 25 µL reactions containing 1X isothermal master mix (OptiGene, Horsham, UK), 0.2 µM forward and reverse outer primers (F3 and B3), 1.6 µM forward and reverse internal primers (FIP and BIP), 0.4 µM forward and reverse loop primers (LF and LB), and 2 µL of RNA template or crude extract. The assays were run in a Genie III LAMP machine (OptiGene) at 65 • C for 45 min. The subsequent melting process from 98 • C to 80 • C was carried out with a ramp rate of −0.05 • C/s.

Sensitivity Test for the Improved YMV RT-LAMP Assay
Purified total RNA (100 ng/µL) from a YMV-infected yam plant (Nig14) was serially diluted in RNA (100 ng/µL) from a YMV-negative plant (Nig15). Ten-fold serial dilutions down to 10 −9 were tested by RT-LAMP and RT-PCR in duplicate assays. The sensitivity of the improved RT-LAMP for detecting YMV from crude RNA extracts was also evaluated. Similar to the purified total RNA, crude RNA extract from Nig14 was diluted ten-fold down to 10 −6 with the crude extract from Nig15 and tested by RT-LAMP.

Indexing of YMV by RT-PCR and RT-LAMP Assays
During routine testing of yam plants for YMV detection, discrepancies were found between the standard RT-PCR test and the RT-LAMP developed by Nkere et al. [23]. Three of six samples that tested positive by RT-PCR ( Figure 1A), namely Gh3, Gh5, and Nig1, and that showed mild symptoms of YMV infection ( Figure 2) tested negative by RT-LAMP ( Figure 1B). The PCR products from Gh5 and Nig1 were sequenced (GenBank accession OQ677014 and OQ677015) and showed 99.1% and 98.9% identity, respectively, to Yam mosaic virus isolate DrCDI1, GenBank AJ305449. In addition to the negative RT-LAMP results with samples Gh3, Gh5, and Nig1, there were also late amplification times (>30 min) obtained for samples Gh1 and Gh2 ( Figure 1B).
Yam mosaic virus isolate DrCDI1, GenBank AJ305449. In addition to the negative RT-LAMP results with samples Gh3, Gh5, and Nig1, there were also late amplification times (>30 min) obtained for samples Gh1 and Gh2 ( Figure 1B).
These unsatisfactory RT-LAMP results prompted the design of new LAMP primers to increase the specificity of the assay for YMV detection. New YMV LAMP primers (YMV1-OPT, Figure 3) were designed and used to test the same samples previously tested by RT-PCR and RT-LAMP using the Nkere et al. [23] primers. The new RT-LAMP using YMV1-OPT primers, subsequently referred to as the improved RT-LAMP test, detected YMV from all six samples within 15 min (Figure 4).   These unsatisfactory RT-LAMP results prompted the design of new LAMP primers to increase the specificity of the assay for YMV detection. New YMV LAMP primers (YMV1-OPT, Figure 3) were designed and used to test the same samples previously tested by RT-PCR and RT-LAMP using the Nkere et al. [23] primers. The new RT-LAMP using YMV1-OPT primers, subsequently referred to as the improved RT-LAMP test, detected YMV from all six samples within 15 min (Figure 4).

Evaluation of Improved YMV RT-LAMP Assay Specificity
RNA extracts from leaves of 14 D. alata plants were tested by RT-PCR for YMV and YMMV, another potyvirus infecting yam. Of these, 9/14 tested positive for YMMV only, 1/14 positive for YMV only, and 1/14 positive for both YMV and YMMV. PCR products of the YMV-positive samples were Sanger-sequenced by the Source BioScience sequencing service (Cambridge, UK), which confirmed the presence of YMV.
The same 14 D. alata RNAs were used to test the specificity of the improved RT-LAMP test. The assay detected YMV from the two YMV-positive samples, DA Nig1 and CTRT127, detected by RT-PCR (Table 4). All other samples were negative for YMV, confirming that there was no cross-reactivity of the YMV1-OPT primers with YMMV or the host plant. Table 4. The detection of YMV from Dioscorea alata samples by RT-PCR and RT-LAMP assays.

Sensitivity of Improved YMV RT-LAMP
The sensitivity of the improved RT-LAMP assay for detecting YMV was compared to RT-PCR using primers by Mumford et al. [26]. RNA obtained from a YMV-infected D. rotundata plant was serially diluted ten-fold down to 10 −9 using RNA from a YMV-negative D. rotundata plant. Each dilution was indexed for YMV by RT-PCR and the improved RT-LAMP assay. YMV positive amplifications were obtained from both assays down to 10 −9 ( Figure 5A,C). The time required to detect YMV in the most dilute sample (10 −9 ) was approximately 32 min ( Figure 5A). Similarly, serially diluted crude RNA extracts derived from incubating one YMV-infected leaf disc in PEG buffer were also tested for YMV via the improved RT-LAMP assay. YMV was detected in the sample RNAs diluted down to 10 −2 ( Figure 5B).

Comparison of Conventional RT-PCR and the New RT-LAMP
Purified total RNAs from 53 leaf samples of D. rotundata and D. alata were tested for YMV using the improved RT-LAMP assay and compared with conventional RT-PCR. A total of 36 samples tested positive for YMV by both tests (Table 5). With RT-LAMP, positive amplification signals were obtained in <26 min compared to >150 min required for RT-PCR. All samples that were negative by RT-LAMP were also negative by RT-PCR. The actin housekeeping gene was targeted by RT-PCR and used as an internal control to confirm the good quality of the RNAs, and YMV-negative results were due to a lack of viral RNA rather than any inhibition of the assay (results not shown).  tive D. rotundata plant. Each dilution was indexed for YMV by RT-PCR and the improved RT-LAMP assay. YMV positive amplifications were obtained from both assays down to 10 −9 ( Figure 5A,C). The time required to detect YMV in the most dilute sample (10 −9 ) was approximately 32 min ( Figure 5A). Similarly, serially diluted crude RNA extracts derived from incubating one YMV-infected leaf disc in PEG buffer were also tested for YMV via the improved RT-LAMP assay. YMV was detected in the sample RNAs diluted down to 10 −2 ( Figure 5B).

Comparison of Conventional RT-PCR and the New RT-LAMP
Purified total RNAs from 53 leaf samples of D. rotundata and D. alata were tested for YMV using the improved RT-LAMP assay and compared with conventional RT-PCR. A total of 36 samples tested positive for YMV by both tests (Table 5). With RT-LAMP, positive amplification signals were obtained in <26 min compared to >150 min required for RT-PCR. All samples that were negative by RT-LAMP were also negative by RT-PCR. The actin housekeeping gene was targeted by RT-PCR and used as an internal control to confirm the good quality of the RNAs, and YMV-negative results were due to a lack of viral RNA rather than any inhibition of the assay (results not shown).

Sequence Identity and Phylogenetic Analysis of YMV Amplicons
The mean pairwise nucleotide identity of Sanger-sequenced PCR products from YMVpositive samples (n = 36) obtained in this study was 97.1%. Nucleotide pairwise comparison of these sequences with YMV CP sequences downloaded from NCBI GenBank (n = 30) revealed 89.2-99% nucleotide identities, higher than the proposed International Committee on Taxonomy of Viruses (ICTV) criterion of <76-77% nucleotide identity for species demarcation of potyvirus CP gene [32,33].
Sequences of YMV isolates obtained from this study were tested for recombination using the Recombination Detection Program (RDP) v.4.101. [34]. No recombination was detected among the YMV sequences. Viruses 2021, 13, x FOR PEER REVIEW 13 of 18

Discussion
This study aimed to strengthen virus diagnostics in the seed yam systems by improving existing diagnostic tests because virus detection is crucial for efficient disease management in clean seed propagation systems, most notably during sanitation programs (review by Diouf et al. [3]). An RT-LAMP assay improved in both its specificity and sensitivity for YMV, one of the most economically damaging yam viruses globally, has in this study been developed to assist in the identifying of virus-free yam planting materials.
The false-negative results obtained by previously reported YMV LAMP primers [23] appear to be due to them having been designed from an alignment of the then available YMV coat protein sequences not fully encompassing diversity in the primer targeted regions. Aligning these primers to 125 YMV coat protein sequences revealed mismatches at the 3 end of the primers (Supplementary Figure S1). Studies have shown that 3 terminal mismatches are detrimental to nucleic acid-based amplifications, resulting in a decreased amplification copy number or complete inhibition of amplification, hence providing falsenegative results [35][36][37][38]. This prompted the need to develop a new YMV LAMP primer set.
The new RT-LAMP primer set, YMV1-OPT, demonstrated higher specificity than the existing RT-LAMP primer set [23], as it detected YMV from samples that tested negative with the existing primer set. This is assumed to be due to the YMV1-OPT primers having been designed to minimise the mismatches to 125 YMV sequences from the Gen-Bank database and inserting degenerate codes at 3 ends where mismatches could not be avoided [39][40][41].
Multiple primer combinations were evaluated during the design and selection process of the YMV1-OPT primers (results not shown). Mismatches were avoided at the 3 ends to the greatest extent; however, where inevitable, nucleotide mismatches with <20% of the aligned sequences in one or two positions were tolerated for the outer and loop primers. Previous studies have shown that mismatches are better tolerated in the outer primers than inner primers [42,43]. This could be because the inner primers initiate the amplification process in LAMP assays, hence the need for specificity. The FIP and BIP primers are a fusion of F1C and F2, and B1C and B2 primers, respectively, with the F2 and B2 regions at the 3 ends and F1C and B1C at the 5 ends. Hence, mismatches at the 3 ends of the F2 and B2 primers were replaced with degenerate codes, while those at the F1C and B1C regions were ignored.
Among the YMV isolates sequenced in this study, 21 clustered with isolates belonging to group III were reported in previous studies [9,24], while 11 clustered with a YMV reference genome, MG711313, from Nigeria [8]. According to the classification described by Bousalem et al. [24], Groups I, II, III, IV, VII, and IX comprise samples collected from the African region. An in-silico analysis of sequence alignments of the YMV1-OPT primers with representative sequences of these African groups indicates that the primers will detect YMV isolates from throughout the West African region. The remaining four YMV isolates, Gh21, Cam3, Tog1, and Nig2, formed four distinct groups, suggesting new phylogenetic groups, perhaps associated with isolates from other parts of the world. However, due to limited resources, only samples obtained from West Africa were used for this study. Further studies will be required to validate the detection of YMV from other yam-growing regions of the world using the YMV1-OPT primers.
The inability of RT-PCR to detect YMV from sample 4 ( Figure 2) that tested positive by RT-LAMP could be due to PCR-inhibitory substances co-extracted with the yam RNA since the yam actin gene was also not detected in the same sample. Mumford and Seal [26] reported that yam tissues contain some PCR-inhibitory substances that could be co-extracted with the RNA. This suggests a higher tolerance of RT-LAMP to inhibitors than RT-PCR, which has also been reported in other studies [23,44,45].
Previous studies have shown that RT-LAMP is at least 100 times more sensitive than RT-PCR [23,46,47]. Nkere et al. [23] reported a sensitivity limit of 1000 fg/µL-100 fg/µL and 0.1 ng/µL-0.01 ng/µL for YMV LAMP primers, and YMV-F3x and YMV-B3x PCR primers, respectively. However, in this study, YMV1-OPT primers and YMV CP 1F and YMV UTR 1R PCR primers [25] both detected YMV from an infected sample down to the lowest dilution tested, 0.1 fg/µL (10 −9 ). However, the differences in sensitivities could be due to variations in the YMV concentration of tested samples. YMV amplification was observed at~32 min in the most dilute RNA sample, 0.1 fg/µL, suggesting that 40 min was sufficient to detect YMV in samples with low virus titre. The improved RT-LAMP assay also detected YMV from crude RNA extracts diluted down to 10 −2 , indicating that this approach can be used for rapid detection and in-field diagnosis of YMV. Overall, these imply that the YMV1-OPT primers are highly sensitive and valuable for laboratory-based and in-field detection of YMV.
Highly specific and sensitive diagnostic tests are required for the reliable diagnosis of plant viruses [22,26,28,48]. However, the development of diagnostic tests is an ongoing task. Diagnostic primers must be reviewed regularly and updated as new virus isolates are reported, as this would prevent false-negative results that might arise from potential diversity in such isolates [24,28,49], as observed in this study. The improved RT-LAMP assay will enhance the specificity of YMV detection in the production of virus-free seed yams in West Africa.

Conclusions
The routine detection of YMV via RT-LAMP using crude RNA extracts offers a significant cost and time-saving alternative to RT-PCR assays being used in the seed systems, which require extensive RNA extraction procedures. Furthermore, amplification products are visualised by monitoring the fluorescence generated by positive samples in real-time, thus reducing the likelihood of post-assay contaminations associated with PCR assays. This study presents an RT-LAMP assay with improved specificity and sensitivity for detecting YMV, which can be implemented at several stages of the seed multiplication process to eliminate YMV-positive samples quickly and cost-effectively. The YMV1-OPT primers designed in this study are being used to develop a ready-to-use YMV commercial kit (YMV1-OPT Isothermal kit, OptiGene). This will reduce the assay preparation time and the risk of contamination from the handling process.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/v15071592/s1, Figure S1: Alignment of YMV coat protein sequences, highlighting the YMV LAMP primers by Nkere et al. [23]; Table S1: Percentage identity matrix of sequence alignment used for phylogenetic analysis.  Data Availability Statement: All sequences have been deposited in GenBank, accession numbers can be found in Table 3.